demonstrating-multi-drug-resistant-mycobacterium-tuberculosis

Demonstrating a Multi-drug Resistant <em>Mycobacterium tuberculosis</em> Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

Akonni Biosystems, Inc.

Cyclin E, a regulator of the cell cycle, affects the behavior of breast-cancer cells. We investigated whether levels of cyclin E in the tumor correlated with survival among patients with breast cancer.

Cyclin E and survival in patients with breast cancer.

The objective of this study was to develop and validate a simple, field-portable, microarray system for monitoring microbial community structure and dynamics in groundwater and subsurface environments, using samples representing site status before acetate injection, during Fe-reduction, in the transition from Fe- to SO(4)(2-)-reduction, and into the SO(4)(2-)-reduction phase. Limits of detection for the array are approximately 10(2)-10(3) cell equivalents of DNA per reaction. Sample-to-answer results for the field deployment were obtained in 4 h. Retrospective analysis of 50 samples showed the expected progression of microbial signatures from Fe- to SO(4)(2-) -reducers with changes in acetate amendment and in situ field conditions. The microarray response for Geobacter was highly correlated with qPCR for the same target gene (R(2) = 0.84). Microarray results were in concordance with quantitative PCR data, aqueous chemistry, site lithology, and the expected microbial community response, indicating that the field-portable microarray is an accurate indicator of microbial presence and response to in situ remediation of a uranium-contaminated site.

Monitoring microbial community structure and dynamics during in situ U(VI) bioremediation with a field-portable microarray analysis system.

This report describes the development and pre-clinical testing of a new, random-access RNA sample preparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porous nucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated with single or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automated QIAcube and manual TruTip methods at 10(6) gene copies influenza A per mL nasopharyngeal aspirate. Influenza A and B amended into nasopharyngeal swabs (in viral transport medium) were detected by real-time RT-PCR at approximately 745 and 370 gene copies per extraction, respectively. RNA extraction efficiency in nasopharyngeal swabs was also comparable to that obtained on an automated QIAcube instrument over a range of input concentrations; the correlation between threshold cycles (or nucleic acid recovery) for TruTip and QIAcube-purified RNA was R(2)>0.99. Preclinical testing of TruTip on blinded nasopharyngeal swab samples resulted in 98% detection accuracy relative to a clinically validated easyMAG extraction method. The physical properties of the TruTip binding matrix and ability to customize its shape and dimensions likewise make it amenable to automation and/or fluidic integration.

Rapid, simple influenza RNA extraction from nasopharyngeal samples.

Ce rapport d&eacute;crit le d&eacute;veloppement des essais pr&eacute;cliniques d&#39;un nouveau vive RNA &eacute;chantillon pr&eacute;paration syst&egrave;me (TruTip) pour les &eacute;chantillons rhinopharyng&eacute;s. Le syst&egrave;me est bas&eacute; sur une matrice de monolithique, poreux nucleic acid binding int&eacute;gr&eacute;e dans l&#39;embout de la pipette a&eacute;rosol r&eacute;sistant et peut &ecirc;tre actionn&eacute; avec pipettes simples ou multicanal. Efficacit&eacute; d&#39;extraction &eacute;quivalentes ont &eacute;t&eacute; obtenues entre QIAcube automatis&eacute;e et m&eacute;thodes manuelles de TruTip &agrave; 10 (6) copies de g&egrave;ne grippe A par aspiration nasopharyng&eacute;e mL. Grippe A et B modifi&eacute; dans le rhinopharynx (en milieu de transport viral) ont &eacute;t&eacute; d&eacute;tect&eacute;s par RT-PCR en temps r&eacute;el &agrave; des copies du g&egrave;ne environ 745 et 370 par extraction, respectivement. L&#39;efficacit&eacute; de la RNA extraction dans le rhinopharynx &eacute;tait &eacute;galement comparable &agrave; celle obtenue sur un instrument automatis&eacute; de QIAcube sur une plage de concentrations d&#39;entr&eacute;e ; la corr&eacute;lation entre les cycles seuil (ou la r&eacute;cup&eacute;ration de l&#39;acide nucl&eacute;ique) pour TruTip et l&#39;ARN purifi&eacute; QIAcube &eacute;tait r &gt; 0,99. Essais pr&eacute;cliniques de TruTip sur des &eacute;chantillons d&#39;&eacute;couvillonnage du rhinopharynx aveugle a entra&icirc;n&eacute; dans la pr&eacute;cision de d&eacute;tection de 98 % par rapport &agrave; une m&eacute;thode d&#39;extraction easyMAG cliniquement valid&eacute;s. Les propri&eacute;t&eacute;s physiques de la matrice de liaison TruTip et de la possibilit&eacute; de personnaliser sa forme et ses dimensions le rendent m&ecirc;me se pr&ecirc;tent &agrave; l&#39;automation et/ou int&eacute;gration fluidique.

Extraction rapide et simple grippe RNA provenant d&#39;&eacute;chantillons rhinopharyng&eacute;s.

Questo rapporto descrive lo sviluppo e il test preclinici di un nuovo accesso casuale RNA campione preparazione sistema (TruTip) per campioni nasofaringei. Il sistema &egrave; basato su una matrice monolitica, porosa dell&#39;acido nucleico associazione incorporata all&#39;interno di un puntale anti-aerosol e pu&ograve; essere azionato con pipette mono o multicanale. Efficienza equivalente di estrazione sono stato ottenuto tra QIAcube automatici e metodi manuali TruTip a 10 copie di gene influenza A per aspirato nasofaringeo mL. Influenza A e B modificato in tamponi nasofaringei (in terreno di trasporto virale) sono state rilevate da RT-PCR in tempo reale a circa 745 e 370 gene copie per estrazione, rispettivamente. L&#39;efficienza di estrazione di RNA in tamponi nasofaringei anche era paragonabile a quella ottenuta su uno strumento automatizzato di QIAcube sopra una gamma di concentrazioni di ingresso; la correlazione tra soglia cicli (o recupero dell&#39;acido nucleico) per TruTip e QIAcube-purificato RNA era R(2) &amp;gt; 0,99. Test preclinici di TruTip su campioni da tampone nasofaringeo accecato ha portato nella precisione di rilevamento del 98% rispetto a un metodo di estrazione easyMAG clinicamente validato. Le propriet&agrave; fisiche della matrice associazione TruTip e possibilit&agrave; di personalizzare la forma e le dimensioni rendono similarmente suscettibili di automazione e/o integrazione fluidici.

Estrazione rapida, semplice influenza RNA da campioni nasofaringei.

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO(3)(-)) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO(3), but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

Profiling in situ microbial community structure with an amplification microarray.

Rapid, simple and efficient influenza RNA purification from clinical samples is essential for sensitive molecular detection of influenza infection. Automation of the TruTip extraction method can increase sample throughput while maintaining performance.

Christopher Knickerbocker

Akonni Biosystems, Inc.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray